NEUTRON STARS ARE A COMPACT ENDPOINT OF THE LIFE OF STARS. THEY LOSE THE BULK OF THEIR ROTATIONAL ENERGY IN THE FORM OF A PULSAR WIND, AN ULTRA-RELATIVISTIC OUTFLOW OF ELECTRONS AND POSITRONS, WHICH PROPERTIES ARE YET ELUSIVE. THIS REQUEST AIMS AT IDENTIFYING WHAT ARE THE CONTROL PARAMETERS BEHIND THE VARIED OBSERVATIONAL PHENOMENOLOGY OF THE PULSED SPECTRA OF NEUTRON STARS, AND TO USE THEM TO PREDICT THEIR X-RAY AND/OR GAMMA-RAY BEHAVIOR SO AS TO GUIDE OBSERVATIONS WITH SATELLITES. THIS PROPOSAL ALSO AIMS AT EXPLORING AND ULTIMATELY OBTAINING A VERSATILE COMBINATION OF HYDRODYNAMICAL, MAGNETO-HYDRODYNAMICAL, AND RADIATIVE SIMULATIONS AND TO APPLY THIS TOOL TO THE MODELLING OF PULSAR WIND NEBULAE. SUCH TOOL, IF VERSATILE ENOUGH TO HANDLE THE FORTHCOMING INCREASE IN THE NUMBER OF DETECTED NEBULAE IN BOTH RADIO AND GAMMA-RAY FREQUENCIES, WILL AT ONCE PROVIDE A FRAMEWORK WHERE TO UNDERSTAND INDIVIDUAL OBSERVATIONS AND ALLOW FOR MEANINGFUL POPULATION ANALYSIS: IT WOULD HAVE AN ENORMOUS IMPACT IN EXTRACTING AND INTERFACING THE PHYSICS OF BOTH THE SQUARE KILOMETER AND THE CHERENKOV TELESCOPE ARRAYS, AT THE TWO ENDS OF THE ELECTROGMAGNETIC SPECTRUM. FOR THE LATTER, IN ADDITION, IT CAN ALSO PLAY A DECISIVE ROLE IN SOLVING FOR THE GAMMA-RAY CONFUSION (THE INABILITY TO SEPARATE THE SOURCES OF RADIATION) GENERATED BY THE DOZENS OF PULSAR WIND NEBULAE TO BE DETECTED IN THE CROWDED GALACTIC CENTER REGION, FOR WHICH WE HERE PROPOSE THE EXPLORATION OF A NEW TECHNIQUE. THIS PROPOSAL HAS A HIGH RISK-HIGH GAIN POTENTIAL: SUCH TOOLS WOULD ALSO HAVE AN IMPACT ON MANY OTHER ASTROPHYSICAL SITUATIONS, IN PARTICULAR, THOSE RELATED WITH PLASMA-MAGNETIC FIELD INTERACTION IN TRANSITIONAL PULSARS AND GAMMA-RAY BINARIES.

THE EMERGENCE OF NEXT-GENERATION SEQUENCING TECHNOLOGIES REPRESENTS A GIANT LEAP TOWARDS THE SYSTEMATIC ANALYSIS OF TRANSCRIPTOMES IN LIVING ORGANISMS. RNA SEQUENCING (RNA-SEQ) HAS BEEN ESTABLISHED AS THE ULTIMATE TOOL TO EXPLORE THE EXPRESSION PROFILE OF CELLS AND TISSUES, PROVIDING A PRECISE, UNBIASED, AND GENOME-WIDE MEASUREMENT OF TRANSCRIPT LEVELS. AN IMPORTANT LIMITATION IN RNA-SEQ STUDIES COMES FROM THE FACT THAT COMPLEX DEVELOPMENTAL AND PHYSIOLOGICAL PROCESSES DEPEND ON THE ACTIVITY OF HETEROGENEOUS CELL POPULATIONS. THIS RESTRICTS CELL-SPECIFIC TRANSCRIPTOMICS TO DISSECTIBLE TISSUES. ALTERNATIVELY, HOMOGENEOUS CELL SUSPENSIONS CAN BE OBTAINED BY CELL SORTING (FACS), BUT THIS METHODOLOGY DEPENDS ON SEVERE CELL DISSOCIATION PROTOCOLS THAT MAY SIGNIFICANTLY ALTER GENE EXPRESSION. THE TRANSLATING RIBOSOME AFFINITY PURIFICATION (TRAP) APPROACH HAS RECENTLY BEEN DEVELOPED AS A POWERFUL TOOL TO OVERCOME THIS LIMITATION. TRAP METHODOLOGY ALLOWS THE CELL-SPECIFIC RECOVERY OF POLYRIBOSOME-ASSOCIATED RNAS BY GENETIC TAGGING OF RIBOSOMES USING CELL AND TISSUE-SPECIFIC PROMOTERS. IN THE PRESENT PROPOSAL WE PLAN TO COMBINE THE TRAP APPROACH WITH ADAPTED ENHANCER TRAP METHODS IN ZEBRAFISH. THE GOAL IS TO CONDUCT A PILOT SCREEN THAT WILL SYSTEMATICALLY GENERATE ZEBRAFISH TRANSGENIC LINES SUITABLE FOR DYNAMIC AND TISSUE-SPECIFIC TRANSCRIPTOMIC INTERROGATION (TRAP-TRAP APPROACH). THE RESULTING COLLECTION OF LINES MAY CONSTITUTE A UNIQUE RESOURCE FOR A BROAD COMMUNITY WORKING IN DEVELOPMENTAL GENETICS, ORGAN PHYSIOLOGY OR DISEASE MODELS. TO MAKE THESE TRANSGENIC LINES, AS WELL AS THE TECHNOLOGY ITSELF, AVAILABLE TO RESEARCHERS OF THE EXPANDING ZEBRAFISH FIELD, WE PLAN TO TRANSFER THE TRAP-TRAP PROTOCOLS TO THE AQUATIC VERTEBRATES PLATFORM, HOSTED IN OUR RESEARCH CENTRE (CABD). THIS CORE FACILITY WILL HAVE THE CAPACITY TO OFFER TECHNOLOGICAL SUPPORT TO USERS INTERESTED IN THE TISSUE-SPECIFIC TRANSCRIPTOMIC ANALYSES PROVIDED BY THE TRAP-TRAP APPROACH.

THE MOLECULAR BASES OF HEPATITIS C VIRUS (HCV) PERSISTENCE ARE LARGELY UNKNOWN. CURRENT MODELS ASSUME THAT THE VIRUS GENETIC MATERIAL REMAINS AS RNA IN THE INFECTED CELL. OUR HYPOTHESIS IS THAT ENDOGENOUS RETROTRANSCRIPTASES ENCODED BY CELLULAR RETROELEMENTS CAN PRODUCE CDNA COPIES OF HCV RNA. WE HAVE HIGH FITNESS HCV POPULATIONS THAT ORIGINATE ELEVATED INTRACELLULAR LEVELS OF HCV RNA THAT SHOULD FAVOR BINDING OF ENDOGENOUS RETROTRANSCRIPTASES AND SYNTHESIS OF HCV CDNA, WITH OR WITHOUT INTEGRATION INTO CELLULAR DNA. WE HAVE ALSO A LARGE COLLECTION OF OLIGONUCLEOTIDE PRIMERS THAT CAN COPY AND AMPLIFY ANY HCV GENOMIC REGION. WE WILL APPLY WALKING TO IDENTIFY THE CELLULAR NUCLEOTIDE SEQUENCE CONTEXT IN WHICH HCV CDNA IS LOCATED. THE PRESENCE OF HCV CDNA COULD EXPLAIN CELLULAR ALTERATIONS THAT FAVOR VIRAL PERSISTENCE, AS WELL AS CELL SEQUELS THAT ARE OBSERVED IN HEPATIC CELLS ONCE THE VIRUS HAS BEEN ELIMINATED BY ANTIVIRAL TREATMENTS. THE PRESENCE OF CDNA WOULD BE A TURNING POINT IN OUR UNDERSTANDING OF PERSISTENT INFECTIONS OF ANIMAL RNA VIRUSES.

THE SPINDLE IS A BIPOLAR ARRAY OF MICROTUBULES TO WHICH THE CHROMOSOMES ATTACH AND THAT FACILITATES AN EQUAL DISTRIBUTION OF THE DUPLICATED GENETIC MATERIAL, ONCE REPLICATED, DURING CELL DIVISION. THE MICROTUBULES THAT CONSTITUTE THIS ARRAY EMANATE FROM STRUCTURES KNOWN AS MICROTUBULE-ORGANIZING CENTERS (MTOCS), WHICH LOCALIZE TO BOTH SPINDLE POLES. THE CELLS INITIALLY BEAR JUST ONE MTOC, WHICH IS DUPLICATED AT THE INITIAL STAGES OF THE CELL CYCLE. REMARKABLY, THERE ARE ONLY THREE OTHER CELLULAR ORGANELLES THAT ARE DUPLICATED DURING THE CELL CYCLE: THE NUCLEUS, THE MITOCHONDRIA AND THE PLASTIDS. INTERESTINGLY, ALL THESE OTHER ORGANELLES HAVE THEIR OWN RELATED GENETIC MATERIAL. THIS HAS LED TO THE PROPOSAL THAT MTOCS MUST ALSO COUNT WITH A SPECIFIC POOL OF ASSOCIATED NUCLEIC ACIDS. RECENT EVIDENCES SUPPORT THAT, IN THE CASE OF THE MTOCS, AND IN CONTRAST TO THE OTHER ORGANELLES DUPLICATED DURING THE CELL CYCLE, IT IS PROBABLY RNA AND NOT DNA MOLECULES THAT COULD BE INHERENTLY LINKED TO THESE STRUCTURES. DESPITE DATA SUSTAINING THE SPECIFIC ASSOCIATION OF RNAS TO MTOCS IS ACCUMULATING, THE FINAL CONFIRMATION OF THE EXISTENCE OF SUCH TRANSCRIPTS, THEIR SHARED CHARACTERISTICS AND THEIR PRECISE CELLULAR FUNCTIONS ARE FASCINATING OPEN QUESTIONS THAT WILL ONLY BE ANSWERED BY FURTHER IDENTIFYING NEW EXAMPLES OF SUCH MOLECULES. ACCORDINGLY, THE MAIN OBJECTIVE OF THIS PROJECT IS TO CARRY OUT THE FIRST GLOBAL SYSTEMATIC ANALYSIS TO IDENTIFY MTOC-ASSOCIATED RNAS IN THE BUDDING YEAST SACCHAROMYCES CEREVISIAE. THE CHARACTERIZATION OF THESE TRANSCRIPTS, THE MAIN MOLECULAR PATHWAYS AND CELLULAR FUNCTIONS IN WHICH THEY PARTICIPATE, AND THE EVALUATION OF THE POSSIBILITY THAT A SPECIFIC POOL OF RNA MOLECULES COULD ASYMMETRICALLY ASSOCIATE TO THE MTOCS IN THIS ORGANISM WILL ALLOW TO UNVEIL NEW MECHANISMS BY WHICH PIVOTAL PROCESSES SUCH AS CELL DIVISION, DIFFERENTIATION AND AGING ARE REGULATED.

THE PRIMARY BENEFIT OF BIOSENSORS IS THE CAPACITY TO GAIN RAPID INFORMATION ON THE PRESENCE OF DIFFERENT ANALYTES. A MAJOR ADVANTAGE OF BIOSENSORS OVER MORE SOPHISTICATED APPROACHES IS THAT IT DOES NOT REQUIRE SOPHISTICATED INSTRUMENTATION. BIOSENSOR DEVELOPMENT IS OF ENORMOUS BIOTECHNOLOGICAL INTEREST AS WITNESSED BY MORE THAN 8000 PATENTS DEPOSITED IN THE WORLD-WIDE PATENT DATABASE THAT HAVE THE TERM ¿BIOSENSOR¿ IN THEIR TITLE. HOWEVER, MOST BIOSENSORS ARE WHOLE CELL BIOSENSORS THAT RELY ON LIFE BACTERIA AND THAT ARE BASED ON CELLULAR ANALYTE RECOGNITION THAT IN TURN TRIGGER CHANGES IN REPORTER GENE TRANSCRIPTION. SUCH BIOSENSORS, ALTHOUGH USEFUL, PRESENT A NUMBER OF DISADVANTAGES RELATED TO THE NEED OF ACTIVE BACTERIA FOR MEASUREMENTS, EFFECTS OF ANALYTES ON BIOSENSOR VIABILITY AND PROBLEMS OF ANALYTE BIOAVAILABILITY. THIS PROJECTS AIMS AT EXPLOITING MAJOR ADVANCES IN BACTERIAL SENSOR RESEARCH SUCH AS THE IDENTIFICATION OF THE LIGAND PROFILE OF AN ENORMOUS NUMBER OF SENSOR DOMAINS AND RESEARCH ON GGDEF BASED SIGNAL TRANSDUCTION SYSTEMS. THIS PROJECT IS BASED ON THE ABUNDANT FAMILY OF PROTEINS CONTAINING A GGDEF DOMAIN AND A SENSOR DOMAIN AT ITS N-TERMINAL EXTENSION. IT HAS BEEN SHOWN THAT LIGAND BINDING TO THE SENSOR DOMAIN ALTERS THE CATALYTIC ACTIVITY OF THE GGDEF DOMAINS. WE ENVISAGE HERE THE CONSTRUCTION OF A NOVEL, ENZYME BASED BIOSENSOR TYPE IN WHICH WELL CHARACTERIZED SENSOR DOMAINS (SUCH AS THOSE FOR AROMATIC CONTAMINANTS OR UREA) ARE FUSED TO THE N-TERMINUS OF GGDEF DOMAINS, SUBSTITUTING THE NATIVE SENSOR DOMAIN. GGDEF DOMAINS CHOSEN HAVE PREVIOUSLY BEEN SHOWN TO BE ACTIVE UNDER IN VITRO CONDITIONS. OUR HYPOTHESIS IS BASED ON THE ASSUMPTION THAT LIGAND BINDING TO THE CHIMERA WILL ALTER GGDEF ACTIVITY THAT CAN BE MONITORED BY THE MALACHITE GREEN BASED ASSAYS THAT PERMITS THE DETECTION OF PICOMOLAR AMOUNTS OF INORGANIC PHOSPHATE. NO INSTRUMENTATION IS REQUIRED TO OBSERVE THE BIOSENSOR OUTPUT SINCE THE PRESENCE OF LIGAND WILL RESULT IN A GREEN COLOR.

PRIONS AND AMYLOID PRION-LIKE AGGREGATES HAVE BEEN DIRECTLY IMPLICATED IN MORE THAN 20 HUMAN DISEASES, AMONG THEM NEURODEGENERATIVE PATHOLOGIES SUCH AS ALZHEIMER¿S, PARKINSON¿S, AND HUNTINGTON¿S DISEASES. PRION PROTEINS ARE SELF-PROPAGATING AND TRANSMISSIBLE PROTEIN ISOFORMS THAT ACCUMULATE AS LARGE STRUCTURE-DRIVEN AGGREGATES, AND IT IS GENERALLY ACCEPTED THAT PRION ACCUMULATION IN THE HUMAN BRAIN IS A DIRECT CAUSE OF NEURONAL DEGENERATION. HOWEVER, APPROPRIATE THERAPEUTIC APPROACHES AND EFFECTIVE TREATMENTS ARE LARGELY LACKING, AND EFFORTS TO PREVENT OR DECREASE THE RATE OF PRION AGGREGATION WITH PEPTIDES HAVE PRODUCED VERY LIMITED RESULTS. HERE WE HYPOTHESIZE THAT, SIMILARLY TO THE OPPOSING TWINS PROMETHEUS AND EPIMETHEUS, ANTIPRIONS COULD ORIGINATE FROM PRION DOMAINS AS QUASI-TWIN STRUCTURES THAT (1) STILL BIND WITH HIGH EFFICIENCY TO PRION AGGREGATES BUT (2) DO NOT TRANSMIT THE PATHOLOGICAL FOLD TO NEWLY RECRUITED MONOMERS, THUS ARRESTING PRION AGGREGATE GROWTH. GIVING SUPPORT TO THIS HYPOTHESIS, PRION MISFOLDING AND AGGREGATION TAKES PLACE IN SUCCESSIVE STEPS OF CONFORMATIONAL CHANGE. HOWEVER, THE STRUCTURAL DETAILS OF THESE TRANSITIONS ARE LARGELY UNKNOWN, MAKING IMPOSSIBLE THE POST HOC DESIGN OF MUTANTS BASED ON PREDICTED STRUCTURAL PROPERTIES. FOR THIS REASON, OUR PROPOSAL IS GROUNDED IN A NON-BIASED APPROACH, AND PLANS TO USE HUMAN PRION SEQUENCES (Aand 946;42 AND and 945;SYN) AS INITIALS SEEDS TO PERFORM (1) AN UNPRECEDENTED, EXTENSIVE AND HIGHLY SENSITIVE RANDOM-MUTAGENESIS BASED SCREEN DESIGNED TO GENERATE AND TEST MORE THAN TEN MILLION INDEPENDENT MUTANT PEPTIDES AS ANTIPRION FACTORS, AND (2) A COMPREHENSIVE FUNCTIONAL SURVEY OF THE ISOLATED ANTIPRION PEPTIDES BY THEIR ABILITY TO COUNTERACT AGGREGATION OF HUMAN PRIONS AND THEIR CONCOMITANT PATHOLOGICAL EFFECTS IN NEURONS.

RNA INTERFERENCE (RNAI) IS A MECHANISM OF GENE REGULATION AND DEFENSE AGAINST EXOGENOUS AND ENDOGENOUS GENETIC ELEMENTS THAT IS TRIGGERED BY THE PRESENCE OF DOUBLE-STRANDED RNA (DSRNA) AND CULMINATES WITH THE SILENCING OF HOMOLOGOUS GENES. IN NEMATODES AND ARTHROPODS, RNAI HAS AWAKED GREAT BIOTECHNOLOGICAL INTEREST SINCE IT HAS BEEN DEMONSTRATED THE POSSIBILITY TO SILENCE GENES THROUGH THE SIMPLE INGESTION OF HOMOLOGOUS DSRNA. IF THE APPROPRIATE GENES ARE CHOSEN, SILENCING LEADS TO A HIGHLY SPECIFIC PEST CONTROL THAT IT IS EASILY ADAPTABLE TO EMERGING PESTS. HOWEVER, ARTHROPODS, UNLIKE NEMATODES, LACK THE RNAI SIGNAL-AMPLIFICATION MECHANISMS BASED ON THE RNA-DEPENDENT RNA POLYMERASE ACTIVITY AND REQUIRE SIGNIFICANT AMOUNTS OF DSRNA TO ACHIEVE AN ADEQUATE PESTICIDAL EFFECT. IN KILLERVIROID WE WANT TO EXPLORE THE POSSIBILITY OF ADAPTING A RECOMBINANT RNA EXPRESSION SYSTEM IN ESCHERICHIA COLI THAT WE HAVE RECENTLY DEVELOPED TO PRODUCE INDUSTRIAL AMOUNTS OF CIRCULAR DOUBLE-STRANDED MINIRNA PESTICIDES. WE WILL EXPLORE WHETHER THE INTRODUCTION OF TWO GROUP I AUTOCATALYTIC INTRONS (THE SECOND OF THEM WITH INTRON-EXON PERMUTATION) ALLOWS THE PRODUCTION OF THE REMARKABLE QUANTITIES OF CIRCULAR DOUBLE-STRANDED MINIRNA PESTICIDES THAT WILL BE NEEDED TO JUMP FROM THE LABORATORY TO THE INDUSTRY. THE RESEARCH WILL FOCUS ON THE GENE SNAKESKIN1 (SSJ1) OF THREE IMPORTANT INSECT PESTS: CERATITIS CAPITATA, DIABROTICA VIRGUIFERA AND TUTA ABSOLUTA, WHICH AFFECT FRUIT, CORN AND TOMATO CROPS, RESPECTIVELY. IF SUCCESSFUL, KILLERVIROID WILL OFFER THE PRODUCTIVE SECTOR OF AGRICULTURAL BIOTECHNOLOGY THE POSSIBILITY OF PRODUCING A NEW GENERATION OF HIGHLY SPECIFIC PESTICIDES, RESPECTFUL WITH THE ENVIRONMENT AND EASILY ADAPTABLE TO EMERGING NEW PESTS OF ARTHROPODS AND NEMATODES.

ASSESSMENT OF CELL PROLIFERATION IS OF CRUCIAL IMPORTANCE IN VARIOUS AREAS OF BIOMEDICAL RESEARCH SUCH AS CANCER OR DRUG DISCOVERY, AND IN MANY BASIC RESEARCH PROJECTS AS WELL. ONE OF THE MOST WIDELY USED TECHNOLOGIES IS TO MEASURE THE INCORPORATION OF ONE OR MORE NUCLEOTIDE ANALOGS IN NEWLY SYNTHESIZED DNA. TO DATE THIS IS ACHIEVED USING IMMUNOLOGICAL PROCEDURES THAT PRESENT SEVERAL DRAWBACKS: (I) NEED OF HARSH TREATMENTS TO DENATURE THE DNA, AFFECTING THE MORPHOLOGY OF CELLS AND TISSUES, AND (II) THE PROCEDURE IS NOT COMPATIBLE WITH THE USE OF OTHER ANTIBODIES, E.G. FOR DETECTING MODIFICATIONS OF CHROMATIN STRUCTURE. THEREFORE, DEVELOPMENT OF NOVEL PROCEDURES IS A MUST IF WE WANT TO INCREASE OUR PORTFOLIO OF TECHNIQUES TO EVALUATE CELL PROLIFERATION IN WHOLE TISSUES AND ORGANS. THE REPLICOLORS PROPOSAL AIMS AT SOLVING THIS PROBLEM BY GENERATING NOVEL AND HIGH VALUE TOOLS USEFUL FOR BASIC EXPERIMENTAL AND BIOTECHNOLOGICAL PURPOSES. IN PARTICULAR, WE PROPOSE TO DEVELOP NEW CHEMICAL REACTIONS TO DETECT MODIFIED NUCLEOTIDES IN NATIVE DNA THAT ARE COMPATIBLE WITH THE CLICK CHEMISTRY, SPECIFICALLY, THE CU(I)-CATALYZED AZIDE-ALKYNE HUISGEN CYCLOADDITION, ALREADY USED IN THIS FIELD. THIS WOULD ALLOW MULTIPLE LABELING OF THE NEWLY SYNTHESIZED DNA AVOIDING THE USE OF HARSH IMMUNOLOGICAL DETECTION AND PREVENTING CELL, TISSUE AND ORGAN STRUCTURE. SUCCESSFUL ACHIEVEMENT OF THIS GENERAL OBJECTIVE NEEDS TO BRING TOGETHER EXPERTISE IN CHEMISTRY AND BIOLOGY FROM TWO DIFFERENT RESEARCH TEAMS. FURTHERMORE, THE MAIN OBJECTIVE IS NOT PART OF OUR CURRENT FUNDED PROJECTS. MORE IMPORTANTLY, THIS PROPOSAL WILL NOT AND CANNOT BE TACKLED IN THE INDIVIDUAL LABORATORIES SEPARATELY UNLESS THEY ARE COORDINATED TOWARDS SETTING UP NEW CHEMICAL REACTIONS AND ADAPTING THEM PROPERLY TO BIOLOGICAL MATERIAL. IT IS RISKY, BUT VERY ATTRACTIVE AND OF HIGH GAIN, AND BASED ON THE EXPERTISE OF THE TWO TEAMS INVOLVED, THERE ARE REASONABLE CHANCES OF SUCCESS.

PLANT VIRUSES CAUSE SIGNIFICANT ECONOMIC LOSSES TO CROPS WORLDWIDE. ALTHOUGH VIRAL RESISTANCE CAN BE ENGINEERED BY PLANT TRANSGENESIS, THIS RAISES PUBLIC CONCERN AND THE EUROPEAN UNION LEGISLATION ON GENETICALLY MODIFIED ORGANISMS IS EXTREMELY RESTRICTIVE. CONVENTIONAL BREEDING SCHEMES REQUIRE IDENTIFICATION OF RESISTANCE SOURCES THAT WILL BE INTROGRESSED TO COMMERCIAL CROPS, WHICH INVOLVES MASSIVE SCREENING OF NATURAL OR MUTANT POPULATIONS. ALTHOUGH GENETIC IDENTIFICATION OF CAUSAL GENES IS GREATLY FACILITATED BY GENOTYPING-BY-SEQUENCING APPROACHES, WE CURRENTLY LACK HIGH-THROUGHPUT PHENOTYPING PROCEDURES FOR VIRAL RESISTANCE, ESPECIALLY WHEN INFECTION SYMPTOMS ARE NOT SEVERE ENOUGH FOR VISUAL DETECTION. OUR PROPOSED SOLUTION IS TO GENERATE ENGINEERED VIRUSES THAT EXPRESS SELECTABLE MARKER GENES. THUS, VIRUS-MEDIATED EXPRESSION OF A D-AMINO ACID OXIDASE GENE WILL ALLOW DUAL, CONDITIONAL NEGATIVE OR POSITIVE SELECTION. APPLICATION OF D-VALINE WILL PERMIT IDENTIFICATION OF INFECTION-RESISTANT PLANTS, WHEREAS IDENTIFICATION OF GAIN-OF-SUSCEPTIBILITY PHENOTYPES WILL BE POSSIBLE USING THE SAME ENGINEERED VIRUS WITH APPLICATION OF D-ALANINE. THE SECOND ALTERNATIVE, IS VERY INNOVATIVE SINCE ALLOWS THE IDENTIFICATION OF GAIN-OF-FUNCTION PHENOTYPES WHICH ARE VERY INTERESTING BUT DIFICULT TO FIND. IN ANOTHER STRATEGY, WE PROPOSE TO EXPRESS CLAVATA3-LIKE GENES BY ENGINEERED VIRUSES. IN ARABIDOPSIS THALIANA, CLAVATA3 OVEREXPRESSION CAUSES A STRONG PHENOTYPE WITH SHOOT MERISTEM ARREST AND IMPAIRED SEED PRODUCTION. WE ANTICIPATE THAT PLANTS SUSCEPTIBLE TO A CLAVATA3-EXPRESSING VIRUS WILL HAVE THE SAME PHENOTYPE, WHEREAS RESISTANT PLANTS WILL SHOW NO DEVELOPMENTAL DEFECTS. THIS SELECTION APPROACH IS COST/TIME-SAVING, SINCE IT DOES NOT REQUIRE SUBSTRATE SUPPLY; MOREOVER, DUE TO CONSERVATION OF CLAVATA3-LIKE GENES IN BOTH MONOCOT AND DICOT SPECIES, WE PREDICT IT COULD BE EASILY EXTENDED TO OTHER PLANT-VIRUS SYSTEMS, INCLUDING CEREAL CROPS.

BIPEDAL LOCOMOTION IS THE ESSENTIAL FEATURE OF HOMININS, SO ESTABLISHING THE MODE OF LOCOMOTION THAT PRECEDED IT IS ESSENTIAL FOR THE KNOWLEDGE OF HUMAN EVOLUTION. HOWEVER, THE HOW, WHERE AND WHY OF ITS ORIGIN ARE QUESTIONS WITH UNCERTAIN ANSWERS. THIS PROJECT PROPOSES TO CHARACTERIZE THE ECOLOGICAL NICHE WHERE HUMAN BIPEDAL LOCOMOTION AROSE THROUGH THE RECONSTRUCTION OF THE LOCOMOTOR REPERTOIRE AND HABITAT USE OF HOMO-PAN LCA. THE AIM IS TO CONTRAST THE MODELS OF LOCOMOTION "SUSPENSION UNDER BRANCHES" VS. "MULTIGRADE: QUADRUPED GENERALIZED PLANTIGRADE ABOVE BRANCHES". FOR THIS PURPOSE, WE PROPOSE TO QUANTIFY THE REPERTOIRE OF ARBOREAL AND TERRESTRIAL LOCOMOTION MODES OF THE PRIMATE SPECIES OF AN AFRICAN TROPICAL FOREST, THE FORMER HYPOTHESIS OF THE ECOSYSTEM ASSUMED FOR THIS LCA. THE CHOSEN PLACE IS THE NATURAL PARK OF MONTE ALEN, EQUATORIAL GUINEA (GE) WHOSE NATIVE FOREST GATHERS EXCEPTIONAL CONDITIONS. THE PROJECT EXPLORES A LOGICAL-ANALYTICAL SCHEME AND A SEQUENCE OF ACTIVITIES TO CAPTURE DATA AND TEST HYPOTHESES ORGANIZED INTO FOUR OBJECTIVES. THE FIRST OBJECTIVE IS THE SEARCH OF FOSSILS THAT PHYSICALLY REPRESENT THE UAC HOMO / PAN IN AN ALMOST UNPRECEDENTED ENVIRONMENT: THE AFRICAN RAIN FOREST. THE DISCOVERY OF ANY PRIMATE FOSSIL IN THIS CONTEXT WOULD BE A TURNING POINT IN WORLD PALEOANTHROPOLOGY. AS A SECOND OBJECTIVE WE PROPOSE THE ANALYSIS OF THE LOCOMOTOR REPERTOIRE AND HABITATS OF THE PRIMATES OF MONTE ALEN. FOR THIS, THE ORIGINAL DATA WILL BE TAKEN IN THE FIELD BY DIRECT OBSERVATION AND FILMING. IN PARALLEL, AND AS A THIRD OBJECTIVE, THE BOTANICAL CHARACTERIZATION IS PURSUED IN THE BIOTOPES OCCUPIED BY THE PRIMATES. WITH THESE DATA, THE FOURTH OBJECTIVE SEEKS TO INFER A MODEL OF LOCOMOTION AND HABITAT USE FOR THE UAC HOMO / PAN (OF ECOLOGICAL NICHE). METHODS OF RECONSTRUCTION OF ANCESTRAL STATES AND THEIR RELATIONSHIP WITH HABITAT VARIABLES WILL BE USED BY MEANS OF ANALYSIS OF COVARIANCE (PLS).